Project Summary/Abstract Fluorescence imaging is having a transformative impact on general medical science with the advent of powerful preclinical techniques such as super-resolution microscopy and single-molecule-tracking, along with emerging clinical procedures such as fluorescence guided surgery and rapid pathology. Each method requires a specific type of fluorescent molecular probe, and in many cases non-optimal probe performance is a major factor that is limiting long term progress and broad impact. Thus, the central objective of this research is to develop greatly improved fluorescent dyes and convert them into molecular probes for important applications in preclinical and clinical imaging. The largest set of projects are based on new fluorescent cyanine heptamethine (Cy7) dyes that emit near-infrared (NIR) light with wavelengths >700 nm and enable imaging of living subjects in the wavelength windows known as NIR I and NIR II (the latter window is also called Short Wavelength Infrared). The new Cy7 dyes have innovative molecular design features that greatly enhance image contrast and permit rapid fine-tuning of probe pharmacokinetic profiles. The near-infrared dyes will be converted into various targeted and bioresponsive molecular probes for: (a) preclinical microscopy, including single-molecule localization microscopy, (b) in vivo fluorescence imaging of animal models of disease, and clinical imaging of patients undergoing surgery, (c) long-term implantable devices that use NIR fluorescence to report health status, and (d) biomedical diagnostics and sequencing methods. Some of these Cy7 dyes will be so stable that they can be incorporated, for the first time, into automated peptide and oligonucleotide synthesis protocols. This will have a major impact on clinical translation since it will allow researchers to reliably make their own Cy7 labeled peptide or oligonucleotide probes on a large scale using a standard synthesizer machine. A second set of projects will develop a new class of fluorescent molecules whose unusual self-threaded structures are reminiscent of entangled peptides. The probes have rigid peptide loops that promote outstanding stability and targeting properties, along with bright deep-red fluorescence that is perfectly suited for advanced intracellular microscopy. An early focus is on molecular probes that spontaneously permeate into living cells and permit selective fluorescence labeling of any intracellular protein containing a polyhistidine sequence (His-tagged protein). The work will create the first deep-red fluorescent molecular probes for rigorous nanoscale imaging of subcellular polar lipids during biomedically important dynamic processes such as viral infection and endosome/exosome trafficking. The utility and versatility of these new fluorescent probes will be demonstrated by collaborations with experts in advanced microscopy. Close coordination with a commercial vendor will ensure that the important fluorescent probes developed by this research quickly become available to all interested investigators.